1. Field of the Invention
The present invention relates to a method of using a compound as an inhibitor for cleavage of amyloid precursor protein (APP) by β-secretase or γ-secretase, wherein the compound binds to the site within β-secretase or γ-secretase recognition and/or cleavage site of APP to block the approach by β-secretase or γ-secretase, while maintaining its activities for other substrates. Further, the present invention relates to inhibitors of amyloid precursor protein processing by β-secretase or γ-secretase, comprising the compound capable of binding to the site within β-secretase or γ-secretase recognition and/or cleavage site of APP. The invention also relates to treating the symptoms of Alzheimer's disease by applying the inhibitors to the person in need thereof.
2. General Background and State of the Art
Alzheimer's disease (AD), the most common cause of dementia in elderly people, is a complex disorder of the central nervous system clinically characterized by a progressive loss of cognitive abilities. Pathological hallmarks of AD are extracellular senile plaques, intracellular neurofibrillary tangles composed of abnormal tau paired helical filaments, loss of neurons, cerebral amyloid angiopathy, and degeneration of cerebrovasculatures in certain areas of the brain (Marti et al., Proc Natl Acad Sci USA 1998; 95(26):15809-15814; Yamada M., Neuropathology 2000; 20(1): 8-22; Yankner B A, Neuron 1996; 16(5):921-932). β-amyloid (Aβ) is the major component of senile plaques and is derived from the amyloid precursor protein by proteolytic cleavage (Vassar et al., Neuron 2000; 27(3): 419-422). Although accumulating evidence suggests that Aβ is a key causative agent of AD (Calhoun et al., Nature 1998; 395(6704):755-756; Hardy et al., Science 1992; 256(5054):184-185; Hsiao et al., Science 1996; 274(5284):99-102; Lewis et al., Science 2001; 293(5534):1487-1491; Schenk et al., Nature 1999; 400(6740):173-177; Sommer B., Curr Opin Pharmacol 2002; 2(1):87-92; Thomas et al., Nature 1996; 380(6570):168-171), the exact mechanism of neuronal degeneration in AD is not clear. However, it is likely that multiple factors are involved in the development of the disease.
Alzheimer's disease (AD) is a progressive neurodegenerative dementia afflicting 1% of the population over age 65. A significant pathological feature, however, is an overabundance of diffuse and compact senile plaques in association and limbic areas of the brain. Although these plaques contain multiple proteins, their cores are composed primarily of β-amyloid, a 40-42 amino acid proteolytic fragment derived from the amyloid precursor protein (Selkoe D J. Cellular and molecular biology of β-amyloid precursor and Alzheimer's disease. In: Prusiner S B, Rosenberg R N, Mauro S D, et al, eds. The molecular and genetic basis of neurological disease. Boston: Butterworth Heinemann Press, 1997:601-602).
APP is a single-transmembrane protein with a 590-680 amino acid long extracellular amino terminal domain and an approximately 55 amino acid cytoplasmic tail which contains intracellular trafficking signals. mRNA from the APP gene on chromosome 21 undergoes alternative splicing to yield eight possible isoforms, three of which (the 695, 751 and 770 amino acid isoforms) predominate in the brain. APP695 is the shortest of the three isoforms and is produced mainly in neurons. Alternatively, APP751, which contains a Kunitz-protease inhibitor (KPI) domain, and APP770, which contains both the KPI domain and an MRC-OX2 antigen domain, are found mostly in non-neuronal glial cells. All three isoforms share the same Aβ, transmembrane and intracellular domains and are thus all potentially amyloidogenic. The normal function of APP is currently unknown, although in neurons it has been demonstrated to be localized in synapses where it may play a role in neurite extension or memory.
APP can undergo proteolytic processing via 2 pathways. Cleavage by α-secretase occurs within the Aβ domain and generates the large soluble N-terminal APPα and a non-amyloidogenic C-terminal fragment. Further proteolysis of this fragment by γ-secretase generates yet other the non-amyloidogenic peptide p3. Alternatively, cleavage of APP by β-secretase occurs at the beginning of the Aβ domain and generates a shorter soluble N-terminus, APPβ, as well as an amyloidogenic C-terminal fragment (C99). Further cleavage of this C-terminal fragment by γ-secretase generates Aβ. Cleavage by γ-secretase or multiple γ-secretases can result in C-terminal heterogeneity of Aβ to generate Aβ40 and Aβ42.
In further detail, APP is trafficked through the constitutive secretory pathway, where it undergoes post-translational processing including a variety of proteolytic cleavage events. APP can be cleaved by three enzymatic activities termed α-, β-, and γ-secretase (FIG. 1). α-secretase cleaves APP at amino acid 17 of the Aβ domain, thus releasing the large amino-terminal fragment sAPPα for secretion. Since α-secretase cleaves within the Aβ domain, this cleavage precludes Aβ formation. Rather, the intracellular carboxy-terminal domain of APP generated by α-secretase cleavage is subsequently cleaved by γ-secretase within the predicted transmembrane domain to generate a 22-24 residue (˜3 kD) fragment termed p3 which is non-amyloidogenic (Sisodia et al., Science; 248:492-5 (1990)). Alternatively, APP can be cleaved by β-secretase to define the amino terminus of Aβ and to generate the soluble amino-terminal fragment APPβ. Subsequent cleavage of the intracellular carboxy-terminal domain of APP by γ-secretase yields full-length Aβ. Carboxy-terminal cleavage of Aβ by γ-secretase results in the generation of multiple peptides, the two most common being 40-amino acid Aβ (Aβ40) and 42-amino acid Aβ (Aβ42). Aβ40 comprises 90-95% of secreted Aβ and is the predominant species recovered from cerebrospinal fluid (Seubert et al., Nature; 359:325-7 (1992)). In contrast, less than 10% of secreted Aβ is Aβ42. Despite the relative paucity of Aβ42 production, Aβ42 is the predominant species found in plaques and is deposited initially (Iwatsubo et al., Neuron; 13:45-53 (1993)), perhaps due to its ability to form insoluble amyloid aggregates more rapidly than Aβ40 (Jarrett et al., Biochemistry; 32:4693-7 (1993); Jarret et al., Cell; 73:1055-89 (1993)).
Aβ has been postulated to be a causal factor in the pathogenesis of AD. The presence of Aβ-containing amyloid plaques is necessary for the neuropathological diagnosis of AD, suggesting that these entities may be involved in the etiology of the disease. Supportive evidence for the causal role of Aβ in AD can be found in patients with Down's syndrome, who often develop AD-like symptoms and pathology after age 40 (Wisniewski et al., Neuron; 35:957-61(1985)). Down's syndrome patients produce elevated APP presumably due to an additional copy of chromosome 21 and exhibit florid AD-like amyloid plaques prior to the onset of other AD symptoms, suggesting that amyloid deposition is an initial event (Giaccone et al., Neurosci Lett; 97:232-8 (1989)). Furthermore, alterations in APP processing have been linked to a subset of familial AD patients (FAD) with autosomal dominant mutations in APP (Goate et al., Nature; 349:704-6 (1991); Citron et al., Nature; 360:672-4 (1992)), presenilin 1 (PS1; 14) and presenilin 2 (PS2; 15).
Given the evidence that altered production of Aβ may be an initial event in the development of AD, much research has focused on understanding the mechanisms by which APP is processed to generate Aβ. The main cleavage pathways appear to be conserved in both neuronal and non-neuronal cells, but the predominant intracellular sites of production and the particular products formed are cell-type dependent. Non-neuronal cells preferentially process APP via α- and γ-secretase cleavage to generate APPα and the non-amyloidogenic fragment p3. Thus, non-neuronal cells are not a significant source of Aβ under normal conditions. However, although non-neuronal cells predominantly utilize α-secretase, neurons do not rely heavily on this pathway and produce very low levels of p3 (Chyung et al., J Cell Bio; 138:671-80 (1997)). Regardless of the cell type, α-secretase cleaves APP constitutively (Sisodia et al., Science; 248:492-5 (1990)) and is thought to occur mainly at the cell surface since APPα cannot be detected intracellularly (Chyumg et al., J Cell Bio; 138:671-80 (1997); Forman et al., J Biol Chem; 272:32247-53(1997)) and cell-surface labeled APP can be recovered as APPα in the medium (Sisodia, Proc Natl Acad Sci USA; 89:6075-9 (1992)). Cleavage by β- and γ-secretases yields Aβ3 and is also a constitutive event, as Aβ can be detected in normal brains in picomolar to nanomolar concentrations (Haass et al., Nature; 359:322-5 (1992); Seubert et al., Nature; 361:260-3 (1993)).
It can be seen that one of the ways to prevent the accumulation of β-amyloid is to prevent β-secretase and/or γ-secretase from cleaving and processing APP. However, secretases are involved in the processing of many important proteins in the organism, and therefore inhibiting secretase activity may cause undesirable side effects. Thus, inactivating β-secretase and/or γ-secretase per se is not an appealing method of preventing APP processing.
Therefore, there is a need in the art to provide a method of treating or preventing Alzheimer's Disease, and in particular inhibiting β-amyloid formation and aggregation. Further, it is desirable to develop compounds that inhibit the processing of APP only without affecting other cellular machinery. Furthermore, design of APP specific inhibitors that can bind to the β-secretase and/or γ-secretase site of APP is desirable to block the approach of these secretases avoiding the processing of other important substrates of these secretases.